Summary: ABCA1 Transporter: In order to determine the structure of the ligand required for efflux from the ABCA1 transporter we have synthesized several synthetic peptides based on the apoA-I amphipathic helix and evaluated the cholesterol efflux from cells in culture. A 37 l amino acid peptide (L-37pA) and a 37 d amino acid peptides(D-37pA) were synthesized and compared with efficiency of apoA-I to efflux cholesterol in cholesterol loaded 293 cell in culture. Both of the peptides were able to effectively efflux cholesterol from the ABCA1 pathways. Several amino acid substitutions of hydrophobic for hydrophilic amino acids were made which established that the amphipathic helix is a necessary prerequisite for efflux from the ABCA1 pathway. A second major protein on HDL is serum amyloid A (SAA), an acute phase protein that associates with HDL. In order to examine the role of SAA in reverse-cholesterol transport, lipid efflux was tested to SAA from HeLa cells before and after transfection with the ABCA1 transporter. ABCA1 expression increased efflux of cholesterol and phospholipid to SAA by 3-fold and 2-fold, respectively. In contrast to apoA-I, SAA also removed lipid without ABCA1; cholesterol efflux from control cells to SAA was 10-fold higher than for apoA-I. Of particular interest was the observation that SAA effluxed cholesterol from Tangier disease fibroblasts and from cells after inhibition of ABCA1 by fixation with paraformaldehyde. These studies establish that SAA can act as a lipid acceptor for ABCA1, but unlike apoA-I, it can also efflux lipid without ABCA1, by a detergent-like extraction process. These results suggest that SAA may play a unique role as an auxiliary lipid acceptor in the removal of lipid from sites of inflammation. ABCA1 Transporter: In our previous studies we established that the ABCA1 transporter, which plays a critical role in the lipidation of extracellular apolipoprotein acceptors, traffics between late endocytic vesicles and the cell surface. The present study provides evidence that ABCA1 in late endocytic vesicles plays a role in cellular lipid efflux. Late endocytic trafficking was defective in Tangier disease fibroblasts that lack functional ABCA1. Consistent with a late endocytic protein trafficking defect, the hydrophobic amine U18666A retained NPC1 in abnormally tubulated, cholesterol-poor, Tangier disease late endosomes, rather than cholesterol-laden lysosomes, as in wild type fibroblasts. Consistent with a lipid trafficking defect, Tangier disease late endocytic vesicles accumulated both cholesterol and sphingomyelin and were immobilized in a perinuclear localization. The excess cholesterol in Tangier disease late endocytic vesicles retained massive amounts of NPC1, which traffics lysosomal cholesterol to other cellular sites. Exogenous apoA-I abrogated the cholesterol-induced retention of NPC1 in wild type but not in Tangier disease late endosomes. Adenovirally mediated ABCA1-GFP expression in Tangier disease fibroblasts corrected the late endocytic trafficking defects and restored apoA-I-mediated cholesterol efflux. ABCA1-GFP expression in wild type fibroblasts also reduced late endosome-associated NPC1, induced a marked uptake of fluorescent apoA-I into ABCA1-GFP-containing endosomes (that shuttled between late endosomes and the cell surface), and enhanced apoA-I-mediated cholesterol efflux. The combined results of this study suggest that ABCA1 converts pools of late endocytic lipids that retain NPC1 to pools that can associate with endocytosed apoA-I, and be released from the cell as nascent high density lipoprotein. ABCG5-ABCG8: The excretion of sterols from the liver and intestine is regulated by the ABCG5 and ABCG8 transporters. In the present studies we have identified a binding site for the orphan nuclear receptor liver receptor homolog-1 (LRH-1) at nt 134-142 of the ABCG5/ABCG8 intergenic region necessary for the acttivity of both the ABCG5 and ABCG8 promoters. Mutating this LRH-1 binding site reduced promoter activity of the human ABCG5/ABCG8 intergenic region more than 7-fold in HepG2 and Caco2 cells. Electrophoretic mobility shift assays with HepG2 nuclear extracts demonstrated specific binding of LRH-1 to the LRH-1 binding motif in the human ABCG5/ABCG8 intergenic region. LRH-1 overexpression increased promoter activity up to 1.6-fold and 3-fold in Caco2 and 293 cells, respectively. In addition, deoxycholic acid repressed the ABCG5 and ABCG8 promoters, consistent with bile acid regulation via the farnesoid X receptor-small heterodimeric partner-LRH-1 pathway. These data demonstrate that LRH-1 is a positive transcription factor for ABCG5 and ABCG8 and, in conjunction with studies on LRH-1 activation of other promoters, identify LRH-1 as a "master regulator" for genes involved in sterol and bile acid secretion from liver and intestine.